Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2014 www.elsevier.com/locate/jbiosc

Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation Xiaoyu Zhou,z Ying Yu,z Jianjun Tao, and Long Yu* State Key Laboratory of Genetic Engineering, Fudan University, 220 Hadan Road, Shanghai 200433, China Received 20 December 2013; accepted 15 March 2014 Available online xxx

Lysozyme acts as an important defensive factor in innate immunity due to its well-recognized bacteriolytic activity. Here we describe the production and performance of human lysozyme-like 6 (LYZL6), a novel human c-type lysozyme homolog. A synthetic codon-optimized cDNA encoding the intact amino acid sequence of LYZL6 was cloned and expressed in Pichia pastoris SMD1168. Bioactive LYZL6 was successfully produced as a single major secreted protein with a molecular weight of 15 kDa, and exhibited bacteriolytic activity against Micrococcus lysodeikticus. The expression conditions were optimized, and the highest expression level of LYZL6 occurred when the recombinant strain was induced with 1.5% methanol under pH 4.5 at 24 C for 96 h. When high cell density fermentation of the recombinant P. pastoris was performed using a fed-batch strategy for totally 125 h in a 30 L fermenter, the dry cell weight and the extracellular lysozyme activity were increased to 116.3 g/L and 2340 U/mL, respectively. The LYZL6 protein concentration was 331 mg/L of fermentation supernatant, and the specific activity of LYZL6 towards M. lysodeikticus was 7069 U/mg. Therefore, we proved that LYZL6 is an antibacterial protein, suggesting a potential application of LYZL6 as an antimicrobial agent, and Pichia expression system for LYZL6 was successful and industrially promising. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: Lysozyme-like; Pichia pastoris; Expression optimization; Fed-batch fermentation; Bacteriolytic activity]

Lysozyme (EC 3.2.1.17), as a defensive effector in innate immunity, plays a crucial role in combating microbial infections by cleaving the b-glycosidic bond between the alternating N-acetylmuramic acid and N-acetylglucosamine residuals in bacterial peptidoglycan, the major bacterial cell wall polymer, thus damaging cell wall and causing bacterial cell lysis (1). Lysozymes distribute widely in nature (2e4) and were categorized into six major types based mainly on their molecular characteristics: chicken-type (c-type), goose-type (g-type), invertebrate-type (itype), plant type, bacterial type and phage type (1,5e8). Among them, the c-type lysozymes have been extensively studied and shown to have a broad distribution, including mammals, birds, reptiles, fishes and insects (1). The well-characterized human lysozyme (H-LYZ), belonging to the c-type lysozyme family, is expressed widely in human body and secreted into a variety of body fluids (9,10). H-LYZ exhibits multiple biological activities, including antimicrobial defense, chemotaxis, protection against human immunodeficiency virus infection and associations with tumors (11e14). Evidence suggested that the catalytic mechanism of c-type lysozymes involved a covalent intermediate in which two essential catalytic residues, Glu-35 and Asp-52, interact with the b-1,4 glycosidic bond of the substrate (15). Human lysozyme-like 6 (LYZL6, also known as LYC1), was a novel c-type lysozyme-like gene identified by our laboratory recently,

* Corresponding author. Tel.: þ86 21 6564 3954; fax: þ86 21 6564 3250. E-mail addresses: [email protected], [email protected] (L. Yu). z The first two authors contributed equally to this work.

and its pattern of expression appeared to be testis/epididymis specific (16). However, earlier systematic investigations using immunohistochemistry methods implied that H-LYZ was not expressed in testicular tissues (9,10). This study was to express LYZL6 in Pichia pastoris and to investigate its putative lysozyme activity. P. pastoris has emerged as a highly efficient heterologous protein expression system in research and industry (17). Unlike the Escherichia coli expression system, which often produces insoluble proteins that lack biological activities when employed as the expression host for eukaryotic proteins, P. pastoris, as a single celled eukaryote, shares the advantages of higher eukaryotic systems such as protein folding and posttranslational modification, while being just as easy to manipulate as E. coli (18). Under the regulation of the strong inducible alcohol oxidase 1 (AOX1) promoter, P. pastoris has produced high levels of recombinant proteins either intracellularly or extracellularly. In the case of high-level extracellular production of recombinant proteins with very low levels of native proteins secreted, the downstream processes were greatly simplified (19). As a yeast, P. pastoris is particularly well-suited for high cell density growth, which has been employed to achieve significantly higher biomass accumulation and usually much higher production level of recombinant proteins, compared to that of traditional shake-flask culture (20). In addition, the cultivation parameters that influence protein expression levels such as pH, temperature, methanol concentration and dissolved oxygen (DO) can be monitored and controlled with convenience in bioreactors (17e19). High cell density fermentation of recombinant P. pastoris is normally separated into three major phases: glycerol batch phase,

1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

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where cells grow on glycerol as carbon source in initial culture, glycerol fed-batch phase, where additional glycerol is fed after exhaustion of the initial glycerol to further increase the cell density, and methanol fed-batch phase, where AOX1 promoter is induced and heterologous protein produced by feeding methanol at an appropriate rate (17,21,22). In this study, codon-optimized LYZL6 was cloned and expressed extracellularly in recombinant P. pastoris. High-level secreted production of LYZL6 was achieved through optimization of expression conditions and high cell density fed-batch fermentation.

4  105% biotin, 0.5% methanol) medium and cultivated at 30 C for 96 h to induce the expression of LYZL6. To maintain the methanol induction, methanol (100%) was added every 24 h to a final concentration of 0.5% throughout the induction period. At the end of induction, culture supernatant was sampled for further studies. P. pastoris SMD1168 transformed with pPIC9K plasmid was used as a negative control.

MATERIALS AND METHODS

High cell density fed-batch fermentation of recombinant P. pastoris Seed culture for fermentation was grown in 1 L of BMGY medium for 20e24 h in shake flasks at 30 C, 250 rpm, until OD600 reached 3e6. The culture was used to inoculate a 30 L jacket fermenter (NC-Bio, Shanghai, China) containing 20 L basal salt medium (0.93 g/L CaSO4, 18.2 g/L K2SO4, 14.9 g/L MgSO4$7H2O, 4.13 g/L KOH, 26.7 mL/L H3PO4, 40.0 g/L glycerol, and 4.0 mL/L PTM1 trace salt solution which consists of 6 g/L CuSO4$5H2O, 0.08 g/L NaI, 2 g/L MnSO4$H2O, 20.2 g/L Na2MoO4, 0.2 g/L H3BO3, 20 g/L CoCl2, 65 g/L FeSO4$7H2O, 0.2 g/L biotin, and 0.5 g/L H2SO4), with a Tophawk Fermentation Control System (NC-Bio) to acquire bioprocess data and a software Bioradar 2.0 (NC-Bio) to monitor the parameters. In order to maintain a constant methanol concentration during the induction phase, a methanol sensor (FC-2000, Super Info. Tech. Co. Ltd., Shanghai, China) was equipped with the fermenter to monitor and control the methanol feeding rate. The important physical and chemical parameters were set according to prior optimization experiments. The temperature was controlled at 30 C during glycerol batch and glycerol fed-batch phases and at 24 C during methanol fed-batch phase, respectively. The pH value of 4.5 was automatically maintained with NH4OH (28% (w/v)). The dissolved oxygen (DO) was kept above 20% of air saturation by adjusting the agitation rate (maximum of 1000 rpm), airflow rate (0.8e1.2 vvm) and adding pure oxygen gas. When initial glycerol in the basal salt medium was exhausted, as indicated by a sudden increase in DO level, the glycerol fed-batch phase was initiated by feeding 50% (w/v) glycerol containing 1.2% PTM1 trace salt solution at the rate of 18.15 mL per h per liter culture broth for a period of 5 h. After that, the feeding of 100% methanol containing 1.2% PTM1 trace salt solution was started at the rate of 3.6 mL per h per liter culture broth for 2e3 h. When the cells were adapted to methanol, as indicated by a stabilized DO level, methanol feeding rate was increased and controlled by the online methanol sensor to maintain the residual methanol concentration at 1.5%. The methanol fedbatch phase lasted for 96 h. During fermentation, samples were collected periodically and stored at 20 C before further analysis for DCW, enzymatic activity, total protein concentration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Microorganisms, plasmids and reagents E. coli TOP10F0 and P. pastoris SMD1168 (His4Mutþ) were purchased from Invitrogen (Carlsbad, CA, USA) and were used as a host strain for DNA manipulation and gene expression, respectively. Plasmid pPIC9K (Invitrogen) was used as the expression vector, and pPIC9 (Invitrogen) was used as an intermediate cloning vector. Micrococcus lysodeikticus, purchased from Chinese Academy of Sciences (Shanghai, China), was used for lysozyme activity assay. Taq DNA polymerase, restriction endonucleases and T4 DNA ligase were purchased from NEB (Ipswich, MA, USA). All chemicals used were biological reagent grade. Design and synthesis of codon-optimized LYZL6 cDNA The usage bias of codons in human and P. pastoris is quite different (23). The cDNA of LYZL6 (GenBank accession no. AY742214) was optimized in favor of P. pastoris expression by the online program DNAWorks and artificially synthesized (Sango, Shanghai, China) to improve the expression level of recombinant LYZL6 in P. pastoris. Enhancement of mRNA stability was considered in addition to codon optimization, and the AT-rich codons were altered to avoid unexpected premature termination. The synthesized 390 base pair (bp) LYZL6 gene shared 72.8% consensus with the wild-type gene (Supplementary material Fig. S1), while the encoding amino acid sequences of wild-type and synthetic cDNA were exactly the same. Construction of the pPIC9k-LYZL6 expression plasmid To achieve secreted expression of LYZL6 with its natural N-terminus, the optimized LYZL6 cDNA was introduced in-frame with the Kex2 signal cleavage site, downstream of the a-factor signal sequence of Saccharomyces cerevisiae in the pPIC9K vector. Vector sequence from XhoI site at bp 1184e1189 to the arginine codon at nucleotides 1193e1195 was rebuilt upstream of the target gene through PCR. The PCR amplification for optimized LYZL6 gene was performed for 30 cycles consisting of 95 C for 30 s, 56 C for 30 s and 72 C for 30 s with two primers, LYZL6F (50 -CCGCTCGAGAAAAGA TCTTTGATTTCTAGATGCGATTTG-30 ) and LYZL6R (50 -CGGAATTCTCATCTCAATCTGCA ACCGG-30 ), flanked by XhoI restriction site on 50 end and EcoRI restriction site on 30 end. The PCR product was purified and digested with XhoI and EcoRI. Since there is another XhoI restriction site outside pPIC9K’s multiple cloning site, the target gene was first inserted into pPIC9, and the resulting plasmid, pPIC9-LYZL6, was transformed into E. coli TOP10F0 and selected on LB plates (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar) containing 100 mg/mL Ampicillin. Positive E. coli transformants were confirmed by direct colony PCR with 50 -AOX1 and 30 -AOX1 primers, 50 GACTGGTTCCAATTGACAAGC-30 and 50 -GCAAATGGCATTCTGACATCC-30 . Positive PCR product was purified, digested with BamHI and EcoRI and ligated to pPIC9K (Fig. 1). The final construct LYZL6-pPIC9K was transformed into E. coli TOP10F0. Transformants were selected as described above, and the recombinant plasmids were isolated from positive transformants, sequenced and used to transform P. pastoris SMD1168 cells. Transformation of P. pastoris and screening of transformants Five microgram of SalI-linearized pPIC9K-LYZL6 was transformed with 80 mL of electrocompetent cells of P. pastoris SMD1168 by electroporation (Gene Pulser Xcell, BioRad, Hercules, CA, USA) at 2.0 kV for 5 ms. After transformation, 0.5 mL of 1 M sorbitol was added into the cuvettes, and 0.1 mL of the suspension was spread onto minimal dextrose agar plates (MD; 13.4 g/L yeast nitrogen base, 10 g/L dextrose, 4  105% biotin, 1.5% agar) to select Hisþ transformants. Multi-copy selection of resistant P. pastoris transformants was carried out by successively replicating the Hisþ transformants onto a series of YPD plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar) containing 0.25, 0.5, 1.0, 2.0 and 4.0 mg/mL Geneticin. Some recombinant clones were selected from the plate containing high concentrations (2.0 and 4.0 mg/mL) of Geneticin, and they were confirmed by colony PCR using genespecific primers. Cultivation of recombinant P. pastoris in shake flasks The selected P. pastoris transformants were inoculated with 3 mL YPD (1% yeast extract, 2% peptone, 2% dextrose) medium in conical tubes and grown at 30 C for 24 h. Then 1 mL of the culture was transferred into 100 mL of BMGY (100 mM potassium phosphate (pH 6.0), 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 4  105% biotin, 1% glycerol) medium in 500 mL shake flasks. After 24 h of growth, the culture was centrifuged at 3000 rpm for 10 min at room temperature to harvest the cells, which were resuspended with 100 mL of BMMY medium (100 mM potassium phosphate (pH 6.0), 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base,

Standardization of cultivation conditions for LYZL6 expression The recombinant clone that expressed the highest LYZL6 activity was chosen for standardization of various expression conditions, such as initial pH value, methanol concentration, induction temperature and period, by maintaining the other parameters constant with the exception of the one being investigated. Based on these experiments, expression of LYZL6 under the standardized conditions was carried out. Samples were taken at set time points and analyzed for lysozyme activity and dry cell weight (DCW).

Determination of DCW The DCW was determined by centrifuging 5 mL of culture broth at 12,000 rpm for 10 min and the pellet was washed with distilled water twice. The cells were dried at 100 C for 24 h, and DCW was calculated. Determination of lysozyme activity The bacteriolytic activity of recombinant LYZL6 was measured by spectrophotometric method using M. lysodeikticus as the substrate. Culture supernatant of 0.1 mL was mixed with 2.5 mL M. lysodeikticus cell suspension (OD450 approximately 0.7) in 50 mM phosphate buffer, pH 6.2. A decrease in absorbance at 450 nm of the mixture caused by the lysis of the bacterial cells was monitored at 25 C. One unit (U) of lysozyme activity was defined as the amount of enzyme reducing OD450 by 0.001 per min. Determination of protein concentration and content Protein concentration was determined by Bradford method using bovine serum albumin (BSA) as the standard (24). SDS-PAGE was performed on a 15% gel according to Laemmli (25), and isolated proteins were visualized by staining with Coomassie Brilliant Blue R-250. The protein bands on SDS-PAGE gels were quantified by a GS-800 Calibrated Densitometer (Bio-Rad). Statistical and computational analysis For each data, replicates from three independent experiments or parallel measurements were measured and the mean values were calculated. Some data were shown as the means  standard deviation (SD). Sequence alignment was done using Clustal W2 program (http://www.ebi. ac.uk) and GeneDoc.

RESULTS Sequence analysis LYZL6 (also known as LYC1) was identified from human by our laboratory recently as a novel c-type lysozymelike gene, which was deposited in GenBank as accession no. AY742214. It is specifically expressed in human testis/epididymis (16). Sequence analysis revealed that as H-LYZ, LYZL6 protein was predicted to contain an N-terminal signal peptide, and sequence alignment of the two proteins identified the presence of eight cysteine residues in LYZL6, which are featured in all members of

Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

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FIG. 1. Construction of the pPIC9K-LYZL6 expression plasmid. S, a-factor signal sequence; HIS4, Pichia wild-type gene coding for histidinol dehydrogenase and used to complement Pichia His4 strains; Kanamycin, Kanamycin resistance gene which confers resistance to Geneticin in Pichia.

the c-type lysozyme family. The two essential catalytic residues of c-type lysozymes, Glu-35 and Asp-52, are also conserved in LYZL6 (Fig. 2). The sequence consensus between mature LYZL6 and HLYZ is 59.5%.

Construction of recombinant LYZL6 expression plasmid Amplification of optimized LYZL6 cDNA with designed PCR primers LYZL6F and LYZL6R resulting in a single band of the expected 413 bp (data not shown), which was cloned into pPIC9

FIG. 2. Amino acid sequence alignment between human lysozyme (H-LYZ) and lysozyme-like 6 (LYZL6). Shaded areas indicate identical/similar residues. The cleavage position of putative signal peptides is indicated. The eight conserved cysteine residues of the c-type lysozyme family are marked by asterisks. Solid triangles indicate positions 35 and 52 of human lysozyme, where the conserved catalytic residues Glu (E) and Asp (D) of c-type lysozymes reside.

Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

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using attached XhoI and EcoRI restriction sites. The plasmid was transformed into E. coli TOP10F0 and the transformants were screened by ampicillin resistance and colony PCR using 50 -AOX1 and 30 -AOX1 primers. Positive PCR product of an expected 880 bp was purified, digested with BamHI and EcoRI and inserted into pPIC9K (Fig. 1). The final construct was subjected to DNA sequencing, which confirmed the correctness of target sequence and its in-frame insertion (data not shown) flush with the Kex2 cleavage site of afactor signal sequence, thus ensuring the secreted expression of natural LYZL6. Screening and expression test of the P. pastoris transformants After the pPIC9K-LYZL6 plasmid had been transformed into P. pastoris SMD1168 host, the vector was integrated into the genomic DNA and the transformants were granted Hisþ phenotype and resistance to Geneticin. Statistically 1e10% of the Hisþ transformants will have multiple inserts. The recombinant P. pastoris capable of resisting higher concentration of Geneticin were inferred to have multiple copies of integrated plasmid and higher expression level of the heterologous protein (26,27). However, the correlation between expression level of the recombinant protein and copy numbers of heterologous gene was more empirical than guaranteed (28), which called for an expression test to determine the actual productivities of the transformants. After screening, only a few transformants could grow in the presence of 2.0 and 4.0 mg/mL Geneticin. We selected 8 highly Geneticin-resistant clones for a flask-scale expression test, and the integration of recombinant plasmid was confirmed by PCR. Four of them were resistant to 4.0 mg/mL Geneticin and the others were resistant to 2.0 mg/mL Geneticin, which were designated as LYZL6-4-1 to LYZL6-4-4 and LYZL6-2-1 to LYZL6-2-4, respectively. SDS-PAGE analysis for the culture supernatants of the tested transformants revealed the expression of approximately 15 kDa LYZL6 (data not shown). Bacteriolytic activity was detected in culture supernatants of all the transformants investigated, and the expression level of LYZL6 appeared to be enhanced with the rise of the transgene dosage (Supplementary material Fig. S2). Among all the transformants tested, SMLYZL6-4-2, which was selected for subsequent experiments, showed the highest lysozyme activity (60.3 U/mL) in its culture supernatant. No lysozyme activity was detected in the supernatant of P. pastoris SMD1168 transformed with pPIC9K vector. Standardization of cultivation conditions for LYZL6 expression We performed a series of flask-scale expression experiments aiming at maximizing LYZL6 production. To investigate the influence of the initial pH on LYZL6 expression, the P. pastoris transformant LYZL6-4-2 was induced in BMMY media of different pH ranging from 3.0 to 7.0 with methanol added to a final concentration of 0.5%, at 30 C for 96 h. Results demonstrated that optimal lysozyme activity expression occurred at initial pH of 4.5, and decreased on both sides (Supplementary material Fig. S3A). The effect of methanol concentration on LYZL6 expression was assessed by inducing selected transformant in BMMY media with initial pH of 4.5, at 30 C for 96 h by adding methanol to different concentrations varying from 0.5% to 3% at a 24 h interval. Maximum expression of lysozyme activity occurred when the culture was induced with 1.5% methanol (Supplementary material Fig. S3B). Then in BMMY with an initial pH of 4.5, the LYZL6-4-2 was induced with 1.5% methanol under different temperatures (22 C, 24 C, 26 C, 28 C and 30 C), and the culture was sampled at a 12 h interval from 48 h to 120 h. Analysis revealed that the highest lysozyme activity was achieved at 24 C for 96 h (Supplementary material Fig. S3C). Based on the experiments above, LYZL6 was expressed by inducing the LYZL6-4-2 strain under the above conditions in

FIG. 3. Time-course profile of LYZL6 production by recombinant P. pastoris LYZL6-4-2 under optimized conditions in 500 mL shake flask. The dry cell weight (circles) and lysozyme activity (triangles) were monitored throughout the process.

500 mL shake flask containing 100 mL of BMGY/BMMY medium. After growth at 30 C in BMGY medium for 24 h, the expression of LYZL6 was induced by changing BMGY to the same volume of BMMY medium. The induction period was carried out under optimal conditions, with an initial pH of 4.5, induction temperature of 24 C and methanol added to a final concentration of 1.5% every 24 h for 96 h. The DCW and enzyme activity was monitored throughout the period. Results demonstrated a rapid increase of DCW during the cultivation in BMGY, which reached 3.3 g/L for 24 h and continued to increase slowly during the induction period, while the lysozyme activity in culture supernatant did not increase apparently until the cells were transferred to BMMY medium (Fig. 3). The DCW and the lysozyme activity finally reached 5.1 g/L and 109 U/mL respectively for 96 h of induction, which was significantly higher than that (60.3 U/mL) expressed with the standard protocol (Invitrogen). Fed-batch fermentation of recombinant P. pastoris to produce LYZL6 Fed-batch fermentation for high-yield production of LYZL6 was carried out in a 30 L bioreactor using a threephase growth protocol (Fig. 4). The glycerol batch phase was first employed for 24 h after inoculation, resulting in a DCW of 23.6 g/ L at the end of the phase when the glycerol fed-batch phase was initiated. After 5 h, the DCW reached 36.4 g/L and the glycerolfeeding was terminated when the methanol fed-batch phase was started to induce the production of LYZL6 by feeding methanol in

FIG. 4. Time-course profile of three-stage fermentation for high-yield production of LYZL6 by recombinant P. pastoris in a 30 L bioreactor. Closed circles, dry cell weight; closed triangles, lysozyme activity.

Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

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a stepwise increasing rate. No apparent lysozyme activity was detected until the methanol fed-batch phase was initiated. After the methanol fed-batch phased for 96 h, the DCW and the lysozyme activity reached final values of 116.3 g/L and 2340 U/ mL, respectively, and the total protein concentration in the final fermentation supernatant was 347.3 mg/L. The DCW and the lysozyme activity in high cell density fermentation increased 22.8 and 21.5 fold respectively, compared with the shake flask cultivation. SDS-PAGE analysis for the time-course samples of the fermentation supernatant demonstrated the gradual increase in the amount of recombinant LYZL6, shown as a single predominant protein band of approximately 15 kDa (Fig. 5), consistent with its expected size of 14.9 kDa. The protein content of LYZL6 was 95.3% of total secreted proteins in the final fermentation supernatant (Fig. 5, lane 5), suggesting that the purity of the produced recombinant LYZL6 was considerably high.

DISCUSSION In earlier study, we identified four c-type lysozyme-like genes, which were highly expressed in the testis/epididymis. Three of them were novel and were named LYZL2, LYZL4, LYZL6 (16), and the other had been reported to encode an intra-acrosomal sperm protein SLLP1 (29). Amino acid sequence analysis further revealed that the two essential catalytic residues of the c-type lysozymes at positions 35 and 52 are conserved in LYZL2 and LYZL6, suggesting that these two proteins may retain bacteriolytic activity, whereas one residual in LYZL4 and two residuals in SLIP1 are replaced, which results in a loss of the lytic activity and the function of SLIP1 involved in sperm-egg binding. We had previously tried to express LYZL6 using the E. coli expression system. However, the LYZL6 was produced as insoluble protein so the downstream processes, including refolding and purification were required to prepare active LYZL6. The expression system of methylotrophic yeast P. pastoris is an excellent cell factory, which is superior to the prokaryotic one in many aspects including stable genomic integration of the expression cassette and

FIG. 5. SDS-PAGE analysis of the recombinant LYZL6 produced in fermentation. Lane M, molecular weight marker (Thermo Scientific); lane 1, uninduced control, sampled at 29 h of fermentation (the end of glycerol fed-batch phase); lanes 2e5, LYZL6 production at 53, 77, 101 and 125 h of fermentation (24, 48, 72 and 96 h after methanol fed-batch phase was initiated), respectively. For each sample, 10 mL of fermentation supernatant was loaded. The arrow indicates the band of expected molecular weight for LYZL6.

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efficient secretion of recombinant proteins, in addition to high cell density culture and posttranslational modification. Secreted expression of enzymatically active LYZL6 was achieved using the S. cerevisiae a-factor signal peptide. The optimized codon composition and screening for recombinant strains with high transgene dosage successfully improved the enzyme activity expressed in culture supernatant. However, the productivity of the expression system depends on optimization of the expression conditions as well as on genetic and physiological traits of the host cell, and it has been reported that the expression levels of heterologous proteins in recombinant P. pastoris could be enhanced by controlling the physical and chemical parameters at optimized levels (30e32). We optimized the expression conditions of pH, methanol addition, induction temperature and period, and the secreted LYZL6 activity under the optimal conditions was 80.8% higher than that expressed with a standard protocol. In P. pastoris expression system, the product formation is generally growth associated, as indicated by the time-course profile of cultivation shown in Figs. 3 and 4. The expression level of LYZL6 in shake-flask culture was relatively low due to the limitations of volume, oxygen transfer, substrate addition and an inability to monitor these factors efficiently. The use of bioreactors is usually preferable, since all of these parameters can be monitored and controlled simultaneously and P. pastoris can grow to a very high cell density, allowing more efficient production of the desired heterologous protein (17), especially when the well-established strategies for P. pastoris fermentation are employed (22,33). In this study, the biomass and the production of LYZL6 were very much improved employing the fed-batch fermentation consisting of glycerol batch phase and fed-batch phase for biomass growth to maximize the cell density, and methanol fed-batch phase where methanol was used both for the induction of target protein and as the carbon source for P. pastoris. Throughout the 96 h of methanol fed-batch phase, the pH of 4.5, temperature of 24 C and residual methanol concentration of 1.5% were maintained automatically, based on the optimization experiments. Finally, bioactive LYZL6 was produced as a single major secreted protein in fermentation supernatant, reaching approximately 331 mg/L, and the calculated specific activity of LYZL6 toward M. lysodeikticus was 7069 U/mg. The high purity of LYZL6 in fermentation supernatant (95.3% of total secreted proteins) generally obviated the purification step. A notable problem for high cell density fermentation is the degradation of recombinant proteins due to release of proteases from host cell and/or dead cell lysis. We chose P. pastoris SMD1168 as the host system, which is a protease-deficient strain, expecting that it might reduce the degree of potential proteolysis. Besides, the relatively low temperature (24 C) and acidic condition (pH 4.5) employed in fermentation would help to limit the protease activity, according to previous studies (34,35). In fact, there was no detectable degradation of recombinant LYZL6 in the final product (Fig. 5). Considering the different expression patterns between H-LYZ, which is widely distributed in a variety of tissues and body fluids, and LYZL6, which is predominantly expressed in the male reproductive system, it is therefore inferred that there may be differences in their substrate specificity and/or suited working conditions. The male genital tract, as a dynamic organ system involved in both endocrine and reproductive functions, has developed an efficient defense mechanism which includes multiple antimicrobial effectors, such as spermine (36), lactoferrin (37), and antimicrobial peptides (38). The confirmed bacteriolytic activity of LYZL6 indicated that the production of LYZL6 in male genital tract may contribute to local antimicrobial defense and play a role in innate immunity of male reproductive system. Further investigation into the effectiveness and specificity of antimicrobial activity of LYZL6 is needed with regard to the natural

Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009

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pathogens, especially those related to reproductive system diseases. Its physiological functions where the local environment is quite different from that of the antimicrobial activity assay remain to be unveiled. However, Pichia expression system for high-yield production of LYZL6 was successful and industrially promising, making it possible to provide enough quantity of protein for future studies and adding to the prospect of it emerging as a candidate for novel antimicrobial agents. In conclusion, a novel human c-type lysozyme LYZL6 was produced as a secreted protein in P. pastoris SMD1168. High-yield production of bioactive LYZL6 was achieved through a combination of codon modification, strain screening, optimization of expression conditions and high cell density fed-batch fermentation. The high yield and purity of LYZL6 in fermentation supernatant generally obviated protein purification thus simplifying enzyme preparation. Its bacteriolytic activity was confirmed against M. lysodeikticus. LYZL6 may contribute to local antimicrobial defense and, with further research, could serve as an alternative bactericide, which could be produced in industrial scale through fermentation. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.03.009.

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Please cite this article in press as: Zhou, X., et al., Production of LYZL6, a novel human c-type lysozyme, in recombinant Pichia pastoris employing high cell density fed-batch fermentation, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.03.009